MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold-induced depolymerization.

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified.

Cap-Gly proteins at microtubule plus ends: is EB1 detyrosination involved?

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids -EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue.

New insights into microtubule elongation mechanisms.

Microtubules are cytoskeletal structures in the cytoplasm of eukaryotic cells, and their highly dynamic properties are essential to perform a wide variety of vital functions in cells. Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin molecules. It has largely been assumed that only tubulin dimers can incorporate into microtubules, and that the chemical state of the nucleotide is crucial for the incorporation.

GDP-tubulin incorporation into growing microtubules modulates polymer stability.

Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data.

Cellular disorders induced by high magnetic fields.

Purpose: To evaluate whether static high magnetic fields (HMFs), in the range of 10-17 T, affect the cytoskeleton and cell organization in different types of mammalian cells, including fibroblasts, epithelial cells, and differentiating neurons.

Materials And Methods: Cells were exposed to HMF for 30 or 60 minutes and subsequently assessed for viability. Cytoskeleton arrays and focal adhesions were visualized using immunofluorescence microscopy.

Identification of proteins binding the native tubulin dimer.

Microtubules play an essential role in eukaryotic cells, where they perform a wide variety of functions. In this paper, we describe the characterization of proteins associated to tubulin dimer in its native form, using affinity chromatography and mass spectrometry. We used an immunoaffinity column with coupled-monoclonal antibody directed against the alpha-tubulin C-terminus.

Microtubule nucleation.

Microtubule nucleation is the process in which several tubulin molecules interact to form a microtubule seed. Microtubule nucleation occurs spontaneously in purified tubulin solutions, and molecular intermediates between tubulin dimers and microtubules have been identified. Microtubule nucleation is enhanced in tubulin solutions by the addition of gamma-tubulin or various gamma-tubulin complexes.

Microtubule nucleation from stable tubulin oligomers.

Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly.

Microtubule dynamics.

Microtubules are fibrous elements in the cytoplasm of eukaryotic cells, where they perform a wide variety of functions. Microtubules are major organizers of the cell interior and are vitally involved in motility events such as chromosome migration during cell division. To fulfill their physiological function, microtubule arrays have to undergo dramatic changes in their spatial arrangement, and this depends to a large extent on the complex and special dynamic properties of the individual polymers.

A reassessment of the factors affecting microtubule assembly and disassembly in vitro.

Current models of microtubule assembly from pure tubulin involve a nucleation phase followed by microtubule elongation at a constant polymer number. Both the rate of microtubule nucleation and elongation are thought to be tightly influenced by the free GTP-tubulin concentration, in a law of mass action-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microtubule length and number concentration during microtubule assembly.

Proliferation of LAMA-84 and LAMA-87 cell lines is modulated by autocrine loops involving M-CSF and TGF-beta.

The erythromegakaryocytic cell line (LAMA-84) and the erythroeosinophilic cell line (LAMA-87) were used to study receptor expression and receptor-mediated response to monocyte/macrophage colony-stimulating factor (M-CSF) and transforming growth factor beta (TGF-beta), two modulators of cell proliferation. As demonstrated by Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), c-fms and M-CSF mRNA were expressed in both cell lines. M-CSF was detected in the supernatant of both cell lines and addition of a neutralizing anti-M-CSF antibody inhibited cell growth.

Effects of retinoic acid on a new human erythromegakaryocytic cell line AP-217.

We have characterized a new human cell line AP-217, derived from the peripheral blood of a patient with chronic myeloid leukemia in blastic crisis. The analysis of cell surface antigens and ploidy showed that AP-217 was an erythro-megakaryocytic cell line. The effects of inducers of differentiation were studied and focused on retinoic acid (RA).

Histone H1(0) expression is restricted to progenitor cells during human hematopoiesis.

The histone H1(0) accumulates in cells with little or no proliferative activity during the terminal phase of differentiation in adult tissues. The hematopoietic cell system is an interesting in vivo model to study the relationship between H1(0) and both the proliferative capacity and differentiation state of cells. Using immunofluorescence techniques, we have analyzed the distribution of histone H1(0) during human hematopoietic differentiation, in normal bone marrow cells and in cell lines representative of cells blocked at early stages of differentiation.

Desmoplakin expression and organization at human umbilical vein endothelial cell-to-cell junctions.

Desmoplakin is an intracellular component of desmosomes which plays a role in the anchorage of intermediate filaments to these structures. We report here that, despite the absence of desmosomes, cultured endothelial cells from human umbilical vein express desmoplakin I and II both at mRNA and protein level. Desmoplakin I/II are found only in the detergent insoluble fraction suggesting that most of the protein is linked to the cytoskeleton.

Selection and characterization of an erythroeosinophilic subclone (LAMA-87) and an eosinophilic subclone (LAMA-88) from the multipotential cell line LAMA-84.

The human leukemic cell line LAMA-84 was established and characterized as an erythromegakaryocytic cell line. In the present study we show that these cells can differentiate in estrone-treated athymic mice and give rise to an erythroeosinophilic cell line (LAMA-87). This new cell line expressed glycoporin A, alpha beta and gamma globin chain mRNA but also eosinophilic peroxidase.

Serum-free medium allows the optimal growth of human megakaryocyte progenitors compared with human plasma supplemented cultures: role of TGF beta.

The growth of human megakaryocyte progenitors from human bone marrow (BM) cells was compared using a methylcellulose semisolid assay supplemented either by normal human plasma or by a serum-free medium. Far better growth of megakaryocyte colonies from CD34+ BM cells stimulated by interleukin 3 (IL-3) and interleukin 6 (IL-6) was observed in serum-free medium compared with human plasma supplemented cultures. These results were confirmed in liquid cultures using the same serum-free medium composition.

Cryopreservation of human megakaryocytic progenitor cells (CFU-MK): influence of culture conditions.

The survival of human megakaryocytic progenitor cells (CFU-MK) after freezing using a two-step cooling technique was studied in a methylcellulose culture system, using different stimulating activities and their combinations. The best growth stimulating activity for CFU-MK was found in plasma from aplastic patients (PAP). PAP was roughly three times more potent than optimal doses of human recombinant interleukin 3 (IL3), itself two times more active than our batch of phytohemagglutinin stimulated leukocyte conditioned medium (PHA-LCM).

Toward an objective classification of cells in the immune system.

The relative abundance of individual proteins shared among clones of lymphocytes provides a meaningful basis for cellular classification. Twelve clones of T cells (obtained by limiting dilution) were analyzed by two-dimensional gel electrophoresis for polypeptide content and then evaluated by the computational technique known as principal component analysis. As a result, relatedness of the clones was established and expressed in terms of taxonomic distances.

Immunophenotype of blast cells in chronic myeloid leukemia.

The immunophenotype of peripheral blood blast cells from 14 patients in the chronic phase of chronic myeloid leukemia (CML) was studied using a panel of monoclonal antibodies (McAb) directed against megakaryocytic, granulomonocytic, erythroid and lymphoid antigenic determinants. The blast cells were enriched by a simple bovine serum albumin (BSA) density-cut separation and cooled in liquid nitrogen. The study was done using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique on the thawed blast cells.

Molecular characterization of an abnormal fibrinogen by two-dimensional electrophoresis.

We examined normal and abnormal fibrinogen (fibrinogen "Grenoble") by two-dimensional gel electrophoresis to obtain data on possible defects at the molecular level. Fibrinogen Grenoble is characterized by an abnormal rate of fibrin monomer aggregation. The electrophoretic analysis revealed the presence of abnormal gamma chains.

A metrological study of autoradiographs from two-dimensional gel electrophoresis.

Samples prepared from a single batch of labeled cells were subjected to two-dimensional gel electrophoresis and autoradiography. Three factors were varied: total quantity of protein, quantity of labeled protein, and exposure time. The mean background absorbance of the film remained identical (about 0.